Fertility requires the formation and maintenance of a healthy germline. Germ cell precursors are specified in the early embryo and later proliferate, undergo meiosis, and differentiate as sperm or oocytes. Throughout development, protective mechanisms ensure that the germline resists outside influences that might alter its fate. This proposal addresses the role of RNA 3? uridylation in regulating gene expression in order to protect germ cell fate and ensure the development of healthy gametes. 3? uridylation is a post- transcriptional modification with context-dependent effects on RNA stability and function. Poly(U) polymerases are nucleotidyl transferases that add uridine residues to the 3? end of RNA molecules. We find that all four poly(U) polymerase proteins in the model nematode, C. elegans, critically effect germline development. In particular, expression of PUP-1 and PUP-2 together prevents aberrant expression of somatic genes, maintains germ cell viability, and ensures gamete formation. Mammalian orthologs, TUT4 (ZCCHC11) and TUT7 (ZCCHC6), are likewise essential for germline and early embryonic development. Strikingly, in the absence of PUP-1 and PUP-2, we find that expression of PUP-3 and PUP-4 contribute to these germline defects. PUP-1 and PUP-2 modify subsets of small RNAs, as do TUT4 and TUT7, however it is unknown if PUP-3 and PUP-4 have this activity. TUT4 and TUT7 also modify mRNA, however it is not known which C. elegans PUPs have this activity. We have an ongoing project to identify PUP-1 and PUP-2 targets. In this proposal, we will perform genetic, protein expression, and RNA sequencing studies to test alternative hypotheses about the developmental roles of pup-3 and pup-4 relative to each other and to pup- 1 and pup-2 (Aim 1). We will perform RNA sequencing experiments to identify uridylated small RNAs and mRNAs in pup-3 and pup-4 single and multiple-mutant strains, including pup-3;pup-1/-2 and pup-4;pup-1/-2 triple mutants (Aim 2). This information will allow us to identify unique and common targets of PUP-3 and PUP-4 activity and correlate loss of uridylation with changes in RNA abundance. We hypothesize that the targets of PUP-3 and PUP-4 activity are different from the targets of PUP-1 and PUP-2 activity. We will test this hypothesis by comparing RNA-sequencing data obtained here with the pup-1 and pup-2 RNA data sets we are now generating (Aim 2.C). Our central hypothesis is that PUP activity modulates the abundance of small RNAs and mRNAs to prevent the loss of germ cell identity, as well as to ensure germline viability and production of healthy gametes.